The VERNALIZATION1 (VRN1) gene encodes a MADS-box transcription factor and plays an important role in the cold-induced transition from the vegetative to reproductive stage. Allelic variability of VRN1 homoeologs has been associated with large differences in flowering time. The aim of this study was to investigate the genetic variability of VRN1 homoeologs (VRN-A1, VRN-B1 and VRN-D1). We performed an in-depth sequence analysis of VRN1 homoeologs in a panel of 105 winter and spring varieties of hexaploid wheat. We describe the novel allele Vrn-B1f with an 836 bp insertion within intron 1 and show its specific expression pattern associated with reduced heading time. We further provide the complete sequence of the Vrn-A1b allele, revealing a 177 bp insertion in intron 1, which is transcribed into an alternative splice variant. Copy number variation (CNV) analysis of VRN1 homoeologs showed that VRN-B1 and VRN-D1 are present in only one copy. The copy number of recessive vrn-A1 ranged from one to four, while that of dominant Vrn-A1 was one or two. Different numbers of Vrn-A1a copies in the spring cultivars Branisovicka IX/49 and Bastion did not significantly affect heading time. We also report on the deletion of secondary structures (G-quadruplex) in promoter sequences of cultivars with more vrn-A1 copies.

Centre of the Region Haná for Biotechnological and Agricultural Research Institute of Experimental Botany of the Czech Academy of Sciences 77900 Olomouc Czech Republic

Department of Cell Biology and Genetics Faculty of Science Palacký University 78371 Olomouc Czech Republic

Department of Plant Developmental Genetics Institute of Biophysics of the Czech Academy of Sciences 61200 Brno Czech Republic

Zobrazit více v PubMed

Dubcovsky J., Dvorak J. Genome plasticity a key factor in the success of polyploid wheat under domestication. Science. 2007;316:1862–1866. doi: 10.1126/science.1143986. PubMed DOI PMC

Beales J., Turner A., Griffiths S., Snape J.W., Laurie D.A. A pseudo-response regulator is misexpressed in the photoperiod insensitive Ppd-D1a mutant of wheat (Triticum aestivum L.) Theor. Appl. Genet. 2007;115:721–733. doi: 10.1007/s00122-007-0603-4. PubMed DOI

Wilhelm E.P., Turner A.S., Laurie D.A. Photoperiod insensitive Ppd-A1a mutations in tetraploid wheat (Triticum durum Desf.) Theor. Appl. Genet. 2009;118:285–294. doi: 10.1007/s00122-008-0898-9. PubMed DOI

Díaz A., Zikhali M., Turner A.S., Isaac P., Laurie D.A. Copy number variation affecting the photoperiod-B1 and vernalization-A1 genes is associated with altered flowering time in wheat (Triticum aestivum) PLoS ONE. 2012;7:e33234. doi: 10.1371/journal.pone.0033234. PubMed DOI PMC

Yan L., Loukoianov A., Tranquilli G., Helguera M., Fahima T., Dubcovsky J. Positional cloning of the wheat vernalization gene VRN1. Proc. Natl. Acad. Sci. USA. 2003;100:6263–6268. doi: 10.1073/pnas.0937399100. PubMed DOI PMC

Trevaskis B., Bagnall D.J., Ellis M.H., Peacock W.J., Dennis E.S. MADS box genes control vernalization-induced flowering in cereals. Proc. Natl. Acad. Sci. USA. 2003;100:13099–13104. doi: 10.1073/pnas.1635053100. PubMed DOI PMC

Yan L., Loukoianov A., Blechl A., Tranquilli G., Ramakrishna W., SanMiguel P., Bennetzen J.L., Echenique V., Dubcovsky J. The wheat VRN2 gene is a flowering repressor down-regulated by vernalization. Science. 2004;303:1640–1644. doi: 10.1126/science.1094305. PubMed DOI PMC

Yan L., Fu D., Li C., Blechl A., Tranquilli G., Bonafede M., Sanchez A., Valarik M., Yasuda S., Dubcovsky J. The wheat and barley vernalization gene VRN3 is an orthologue of FT. Proc. Natl. Acad. Sci. USA. 2006;103:19581–19586. doi: 10.1073/pnas.0607142103. PubMed DOI PMC

Tamaki S., Matsuo S., Hann L.W., Yokoi S., Shimamoto K. Hd3a protein is a mobile flowering signal in rice. Science. 2007;316:1033–1036. doi: 10.1126/science.1141753. PubMed DOI

Chouard P. Vernalization and its relations to dormancy. Annu. Rev. Plant Physiol. 1960;11:191–238. doi: 10.1146/annurev.pp.11.060160.001203. DOI

Alonso-Peral M.M., Oliver S.N., Casao M.C., Greenup A.A., Trevaskis B. The promoter of the cereal VERNALIZATION1 gene is sufficient for transcriptional induction by prolonged cold. PLoS ONE. 2011;6:e29456. doi: 10.1371/journal.pone.0029456. PubMed DOI PMC

Fu D., Szucs P., Yan L., Helguera M., Skinner J.S., Von Zitzewitz J., Hayes P.M., Dubcovsky J. Large deletions within the first intron in VRN-1 are associated with spring growth habit in barley and wheat. Mol. Genet. Genom. 2005;273:54–65. doi: 10.1007/s00438-004-1095-4. PubMed DOI

Xiao J., Xu S., Li C., Xu Y., Xing L., Niu Y., Huan Q., Tang Y., Zhao C., Wagner D., et al. O-GlcNAc-mediated interaction between VER2 and TaGRP2 elicits TaVRN1 mRNA accumulation during vernalization in winter wheat. Nat. Commun. 2014;5:4572. doi: 10.1038/ncomms5572. PubMed DOI PMC

Kippes N., Guedira M., Lin L., Alvarez M.A., Brown-Guedira G.L., Dubcovsky J. Single nucleotide polymorphisms in a regulatory site of VRN-A1 first intron are associated with differences in vernalization requirement in winter wheat. Mol. Genet. Genom. 2018;293:1231–1243. doi: 10.1007/s00438-018-1455-0. PubMed DOI PMC

Yan L., Helguera A.M., Kato A.K., Fukuyama A.S., Sherman J., Dubcovsky A.J. Allelic variation at the VRN-1 promoter region in polyploid wheat. Theor. Appl. Genet. 2004;109:1677–1686. doi: 10.1007/s00122-004-1796-4. PubMed DOI

Zhang X., Gao M., Wang S., Chen F., Cui D. Allelic variation at the vernalization and photoperiod sensitivity loci in Chinese winter wheat cultivars (Triticum aestivum L.) Front. Plant Sci. 2015;6:470. doi: 10.3389/fpls.2015.00470. PubMed DOI PMC

Santra D.K., Santra M., Allan R.E., Campbell K.G., Kidwell K.K. Genetic and molecular characterization of vernalization genes Vrn-A1, Vrn-B1, and Vrn-D1 in spring wheat germplasm from the pacific northwest region of the U.S.A. Plant Breed. 2009;128:576–584. doi: 10.1111/j.1439-0523.2009.01681.x. DOI

Zhang J., Wang Y., Wu S., Yang J., Liu H., Zhou Y. A single nucleotide polymorphism at the Vrn-D1 promoter region in common wheat is associated with vernalization response. Theor. Appl. Genet. 2012;125:1697–1704. doi: 10.1007/s00122-012-1946-z. PubMed DOI

Milec Z., Tomková L., Sumíková T., Pánková K. A new multiplex PCR test for the determination of Vrn-B1 alleles in bread wheat (Triticum aestivum L.) Mol. Breed. 2012;30:317–323. doi: 10.1007/s11032-011-9621-7. DOI

Shcherban A.B., Efremova T.T., Salina E.A. Identification of a new Vrn-B1 allele using two near-isogenic wheat lines with difference in heading time. Mol. Breed. 2012;29:675–685. doi: 10.1007/s11032-011-9581-y. DOI

Steinfort U., Trevaskis B., Fukai S., Bell K.L., Dreccer M.F. Vernalisation and photoperiod sensitivity in wheat: Impact on canopy development and yield components. Field Crops Res. 2017;201:108–121. doi: 10.1016/j.fcr.2016.10.012. DOI

Muterko A., Balashova I., Cockram J., Kalendar R., Sivolap Y. The new wheat vernalization response allele Vrn-D1s is caused by DNA transposon insertion in the first intron. Plant Mol. Biol. Rep. 2015;33:294–303. doi: 10.1007/s11105-014-0750-0. DOI

Springer N.M., Ying K., Fu Y., Ji T., Yeh C.T., Jia Y., Wu W., Richmond T., Kitzman J., Rosenbaum H., et al. Maize inbreds exhibit high levels of copy number variation (CNV) and presence/absence variation (PAV) in genome content. PLoS Genet. 2009;5:e1000734. doi: 10.1371/journal.pgen.1000734. PubMed DOI PMC

McHale L.K., Haun W.J., Xu W.W., Bhaskar P.B., Anderson J.E., Hyten D.L., Gerhardt D.J., Jeddeloh J.A., Stupar R.M. Structural variants in the soybean genome localize to clusters of biotic stress-response genes. Plant Physiol. 2012;159:1295–1308. doi: 10.1104/pp.112.194605. PubMed DOI PMC

Bai Z., Chen J., Liao Y., Wang M., Liu R., Ge S., Wing R.A., Chen M. The impact and origin of copy number variations in the Oryza species. BMC Genom. 2016;17:261. doi: 10.1186/s12864-016-2589-2. PubMed DOI PMC

Yu P., Wang C., Xu Q., Feng Y., Yuan X., Yu H., Wang Y., Tang S., Wei X. Detection of copy number variations in rice using array-based comparative genomic hybridization. BMC Genom. 2011;12:6–13. doi: 10.1186/1471-2164-12-372. PubMed DOI PMC

Muterko A., Salina E. VRN1-ratio test for polyploid wheat. Planta. 2019;250:1955–1965. doi: 10.1007/s00425-019-03279-z. PubMed DOI

Guedira M., Xiong M., Hao Y.F., Johnson J., Harrison S., Marshall D., Brown-Guedira G. Heading date QTL in winter wheat (Triticum aestivum L.) coincide with major developmental genes VERNALIZATION1 and PHOTOPERIOD1. PLoS ONE. 2016;11:e0154242. doi: 10.1371/journal.pone.0154242. PubMed DOI PMC

Würschum T., Boeven P.H.G.G., Langer S.M., Longin C.F.H., Leiser W.L. Multiply to conquer: Copy number variations at Ppd-B1 and Vrn-A1 facilitate global adaptation in wheat. BMC Genet. 2015;16:96. doi: 10.1186/s12863-015-0258-0. PubMed DOI PMC

Kippes N., Debernardi J.M., Vasquez-Gross H.A., Akpinar B.A., Budak H., Kato K., Chao S., Akhunov E., Dubcovsky J. Identification of the VERNALIZATION 4 gene reveals the origin of spring growth habit in ancient wheats from South Asia. Proc. Natl. Acad. Sci. USA. 2015;112:E5401–E5410. doi: 10.1073/pnas.1514883112. PubMed DOI PMC

Li G., Yu M., Fang T., Cao S., Carver B.F., Yan L. Vernalization requirement duration in winter wheat is controlled by TaVRN-A1 at the protein level. Plant J. 2013;76:742–753. doi: 10.1111/tpj.12326. PubMed DOI PMC

Košner J., Pánková K. Vernalization response of some winter wheat cultivars (Triticum aestivum L.) Czech J. Genet. Plant Breed. 2002;38:97–103. doi: 10.17221/6242-CJGPB. DOI

Khan A.R., Enjalbert J., Marsollier A.C., Rousselet A., Goldringer I., Vitte C. Vernalization treatment induces site-specific DNA hypermethylation at the VERNALIZATION-A1 (VRN-A1) locus in hexaploid winter wheat. BMC Plant Biol. 2013;13:209. doi: 10.1186/1471-2229-13-209. PubMed DOI PMC

Strejčková B., Čegan R., Pecinka A., Milec Z., Šafář J. Identification of polycomb repressive complex 1 and 2 core components in hexaploid bread wheat. BMC Plant Biol. 2020;20:175. doi: 10.1186/s12870-020-02384-6. PubMed DOI PMC

Diallo A.O., Ali-Benali M.A., Badawi M., Houde M., Sarhan F. Expression of vernalization responsive genes in wheat is associated with histone H3 trimethylation. Mol. Genet. Genom. 2012;287:575–590. doi: 10.1007/s00438-012-0701-0. PubMed DOI

Svačina R., Karafiátová M., Malurová M., Serra H., Vítek D., Endo T.R., Sourdille P., Bartoš J. Development of Deletion Lines for Chromosome 3D of Bread Wheat. Front. Plant Sci. 2020;10:1756. doi: 10.3389/fpls.2019.01756. PubMed DOI PMC

Guiblet W.M., Cremona M.A., Cechova M., Harris R.S., Kejnovská I., Kejnovsky E., Eckert K., Chiaromonte F., Makova K.D. Long-read sequencing technology indicates genome-wide effects of non-B DNA on polymerization speed and error rate. Genome Res. 2018;28:1767–1778. doi: 10.1101/gr.241257.118. PubMed DOI PMC

Muterko A., Salina E. Origin and distribution of the VRN-A1 exon 4 and exon 7 haplotypes in domesticated wheat species. Agronomy. 2018;8:156. doi: 10.3390/agronomy8080156. DOI

Pidal B., Yan L., Fu D., Zhang F., Tranquilli G., Dubcovsky J. The CArG-box located upstream from the transcriptional start of wheat vernalization gene VRN1 is not necessary for the vernalization response. J. Hered. 2009;100:355–364. doi: 10.1093/jhered/esp002. PubMed DOI

Shcherban A.B., Strygina K.V., Salina E.A. VRN-1 gene- associated prerequisites of spring growth habit in wild tetraploid wheat T. dicoccoides and the diploid A genome species. BMC Plant Biol. 2015;15:94. doi: 10.1186/s12870-015-0473-x. PubMed DOI PMC

Loukoianov A. Regulation of VRN-1 vernalization genes in normal and transgenic polyploid wheat. Plant Physiol. 2005;138:2364–2373. doi: 10.1104/pp.105.064287. PubMed DOI PMC

Emtseva M.V., Efremova T.T., Arbuzova V.S. The influence of Vrn-B1a and Vrn-B1c alleles on the length of developmental phases of substitution and near-isogenic lines of common wheat. Russ. J. Genet. 2013;49:545–552. doi: 10.1134/S1022795413050050. PubMed DOI

Maizels N., Gray L.T. The G4 Genome. PLoS Genet. 2013;9:e1003468. doi: 10.1371/journal.pgen.1003468. PubMed DOI PMC

Muterko A., Kalendar R., Salina E. Novel alleles of the VERNALIZATION1 genes in wheat are associated with modulation of DNA curvature and flexibility in the promoter region. BMC Plant Biol. 2016;16:65–81. doi: 10.1186/s12870-015-0691-2. PubMed DOI PMC

Cagirici H.B., Sen T.Z. Genome-wide discovery of G-quadruplexes in wheat: Distribution and putative functional roles. G3 Genes Genomes Genet. 2020;10:2021–2032. doi: 10.1534/g3.120.401288. PubMed DOI PMC

Lago S., Nadai M., Cernilogar F.M., Kazerani M., Domíniguez Moreno H., Schotta G., Richter S.N. Promoter G-quadruplexes and transcription factors cooperate to shape the cell type-specific transcriptome. Nat. Commun. 2021;12:3885. doi: 10.1038/s41467-021-24198-2. PubMed DOI PMC

Wu F., Niu K., Cui Y., Li C., Lyu M., Ren Y., Chen Y., Deng H., Huang L., Zheng S., et al. Genome-wide analysis of DNA G-quadruplex motifs across 37 species provides insights into G4 evolution. Commun. Biol. 2021;4:98. doi: 10.1038/s42003-020-01643-4. PubMed DOI PMC

Spiegel J., Cuesta S.M., Adhikari S., Hänsel-Hertsch R., Tannahill D., Balasubramanian S. G-quadruplexes are transcription factor binding hubs in human chromatin. Genome Biol. 2021;22:117:1–117:15. doi: 10.1186/s13059-021-02324-z. PubMed DOI PMC

Cagirici H.B., Budak H., Sen T.Z. Genome-wide discovery of G-quadruplexes in barley. Sci. Rep. 2021;11:7876. doi: 10.1038/s41598-021-86838-3. PubMed DOI PMC

Chen Y., Carver B.F., Wang S., Zhang F., Yan L. Genetic loci associated with stem elongation and winter dormancy release in wheat. Theor. Appl. Genet. 2009;118:881–889. doi: 10.1007/s00122-008-0946-5. PubMed DOI

Zhu J., Pearce S., Burke A., See D.R., Skinner D.Z., Dubcovsky J., Garland-Campbell K. Copy number and haplotype variation at the VRN-A1 and central FR-A2 loci are associated with frost tolerance in hexaploid wheat. Theor. Appl. Genet. 2014;127:1183–1197. doi: 10.1007/s00122-014-2290-2. PubMed DOI PMC

Pugsley A.T. A genetic analysis of the spring-winter habit of growth in wheat. Aust. J. Agric. Res. 1971;22:21–31. doi: 10.1071/AR9710021. DOI

Esquerré T., Laguerre S., Turlan C., Carpousis A.J., Girbal L., Cocaign-Bousquet M. Dual role of transcription and transcript stability in the regulation of gene expression in Escherichia coli cells cultured on glucose at different growth rates. Nucleic Acids Res. 2014;42:2460–2472. doi: 10.1093/nar/gkt1150. PubMed DOI PMC

Jędrak J., Ochab-Marcinek A. Influence of gene copy number on self-regulated gene expression. J. Theor. Biol. 2016;408:222–236. doi: 10.1016/j.jtbi.2016.08.018. PubMed DOI

Zhou J., Lemos B., Dopman E.B., Hartl D.L. Copy-number variation: The balance between gene dosage and expression in Drosophila melanogaster. Genome Biol. Evol. 2011;3:1014–1024. doi: 10.1093/gbe/evr023. PubMed DOI PMC

Schuster-Böckler B., Conrad D., Bateman A. Dosage sensitivity shapes the evolution of copy-number varied regions. PLoS ONE. 2010;5:20894. doi: 10.1371/journal.pone.0009474. PubMed DOI PMC

Zhao F., Wang Y., Zheng J., Wen Y., Qu M., Kang S., Wu S., Deng X., Hong K., Li S., et al. A genome-wide survey of copy number variations reveals an asymmetric evolution of duplicated genes in rice. BMC Biol. 2020;18:73. doi: 10.1186/s12915-020-00798-0. PubMed DOI PMC

Akhunova A.R., Matniyazov R.T., Liang H., Akhunov E.D. Homoeolog-specific transcriptional bias in allopolyploid wheat. BMC Genom. 2010;11:505. doi: 10.1186/1471-2164-11-505. PubMed DOI PMC

Ramírez-González R.H., Borrill P., Lang D., Harrington S.A., Brinton J., Venturini L., Davey M., Jacobs J., van Ex F., Pasha A., et al. The transcriptional landscape of polyploid wheat. Science. 2018;361:eaar6089. doi: 10.1126/science.aar6089. PubMed DOI

Voss-Fels K.P., Robinson H., Mudge S.R., Richard C., Newman S., Wittkop B., Stahl A., Friedt W., Frisch M., Gabur I., et al. VERNALIZATION1 modulates root system architecture in wheat and barley. Mol. Plant. 2018;11:226–229. doi: 10.1016/j.molp.2017.10.005. PubMed DOI

Nishida H., Yoshida T., Kawakami K., Fujita M., Long B., Akashi Y., Laurie D.A., Kato K. Structural variation in the 5′ upstream region of photoperiod-insensitive alleles Ppd-A1a and Ppd-B1a identified in hexaploid wheat (Triticum aestivum L.), and their effect on heading time. Mol. Breed. 2013;31:27–37. doi: 10.1007/s11032-012-9765-0. DOI

Untergasser A., Cutcutache I., Koressaar T., Ye J., Faircloth B.C., Remm M., Rozen S.G. Primer3-new capabilities and interfaces. Nucleic Acids Res. 2012;40:e115. doi: 10.1093/nar/gks596. PubMed DOI PMC

Vrana J., Kubalakova M., Simkova H., Cihalikova J., Lysak M.A., Dolezel J. Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.) Genetics. 2000;156:2033–2041. doi: 10.1093/genetics/156.4.2033. PubMed DOI PMC

Giorgi D., Farina A., Grosso V., Gennaro A., Ceoloni C., Lucretti S. FISHIS: Fluorescence in situ hybridization in suspension and chromosome flow sorting made easy. PLoS ONE. 2013;8:e57994. doi: 10.1371/journal.pone.0057994. PubMed DOI PMC

Bolger A.M., Lohse M., Usadel B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. doi: 10.1093/bioinformatics/btu170. PubMed DOI PMC

Li H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv. 20131303.3997

Li H., Handsaker B., Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R. The sequence alignment/map format and samtools. Bioinformatics. 2009;25:2078–2079. doi: 10.1093/bioinformatics/btp352. PubMed DOI PMC

Bankevich A., Nurk S., Antipov D., Gurevich A.A., Dvorkin M., Kulikov A.S., Lesin V.M., Nikolenko S.I., Pham S.O.N., Prjibelski A.D., et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012;19:455–477. doi: 10.1089/cmb.2012.0021. PubMed DOI PMC

Robinson J.T., Thorvaldsdóttir H., Wenger A.M., Zehir A., Mesirov J.P. Variant review with the integrative genomics viewer. Cancer Res. 2017;77:e31–e34. doi: 10.1158/0008-5472.CAN-17-0337. PubMed DOI PMC

Blake V.C., Woodhouse M.R., Lazo G.R., Odell S.G., Wight C.P., Tinker N.A., Wang Y., Gu Y.Q., Birkett C.L., Jannink J.L., et al. GrainGenes: Centralized small grain resources and digital platform for geneticists and breeders. Database. 2019;2019:baz065. doi: 10.1093/database/baz065. PubMed DOI PMC

Bikandi J., Millán R.S., Rementeria A., Garaizar J. In silico analysis of complete bacterial genomes: PCR, AFLP-PCR and endonuclease restriction. Bioinformatics. 2004;20:798–799. doi: 10.1093/bioinformatics/btg491. PubMed DOI

Ivaničová Z., Valárik M., Pánková K., Trávníčková M., Doležel J., Šafář J., Milec Z. Heritable heading time variation in wheat lines with the same number of Ppd-B1 gene copies. PLoS ONE. 2017;12:e0183745. doi: 10.1371/journal.pone.0183745. PubMed DOI PMC

Wild emmer wheat, the progenitor of modern bread wheat, exhibits great diversity in the VERNALIZATION1 gene

Contemplation on wheat vernalization